The objective of this application is to understand osteogenic mechanisms of stem cells from human exfoliated deciduous teeth (SHED) as a basis for the development of therapeutic technologies for repairing orofacial bone defects. Autologous grafts from long bones that are commonly used to repair orofacial bone defects often result in unfavorable outcomes, which may be due, in part, to the fact that orofacial and long bones originate from neural crest cells and mesoderm, respectively, and orofacial mesenchymal stem cells show a distinctive differentiation trait from long bone mesenchymal stem cells. Previously, we showed that SHED are capable of differentiating into odontoblasts, adipocytes, and neural cells. However, one of the most distinct characteristics of SHED is their strong osteogenic capacity when transplanted into immunocompromised mice. Our preliminary studies demonstrated that SHED could be utilized to repair critical-size parietal defects in mice, offering an attractive stem cell resource for orofacial bone regeneration. Interestingly, our preliminary studies showed also that SHED has a gene expression profile distinct from that of bone marrow mesenchymal stem cells (BMMSCs), which correspond to the fact that SHED-generated bone structure lacks associated bone marrow elements, as seen in BMMSC transplants. Our hypothesis is that SHED, derived from neural crest cells, possesses a unique osteogenic trait and may be an optimal stem cell resource for repairing orofacial bone defects. In this application, we will examine whether a sub-population of SHED purified according to their surface molecules show superior differentiation and tissue regeneration capacities. We will characterize the distinctive osteogenic trait of SHED, such as how BMP-2 and bFGF regulate SHED-mediated osteogenesis. On the basis of our novel findings on the maintenance of BMMSCs function by telomerase, we examine how to use growth factors to activate telomerase activity in SHED for improving their tissue regeneration capacity. Finally, we develop optimal conditions to expand minipig SHED and utilize them for autologous transplantation to repair parietal defects, which is a necessary pre-clinical step to examine any efficacy and potential challenges before conducting a human SHED trial. Collectively, novel findings from our proposed studies will provide a molecular basis for understanding SHED-mediated bone formation and have important impacts in orofacial bone regeneration.